High throughput sequencing technologies requiring higher quality and larger nucleic acid samples to analyze the existing microbial flora in an environmental sample are rapidly overtaking previously existing analytical methods in the field. The inability to recover nucleic acids of sufficient quantity and quality from iron oxide complexed clay limits nucleic acid based assessment of many environments. When comparing nucleic acid extraction procedures, a higher nucleic acid yield can indicate low sampling bias and thus a more complete assessment of the microbial community. See Cullen and Hirsch, Soil Biology & Biochemistry. (1998) 30: 983-993; Frostegard et al., Appl. Environ. Microbiol. (1999) 65: 5409-5420. However, nucleic acid isolation methods differ in their ability to recover nucleic acid from cells within a sample due to loss to adsorption of nucleic acids by cations present on the surface of clay particles in an environmental sample, making it difficult to judge microbial community coverage on the basis of nucleic acid yield.
For iron rich clays, DNA and RNA yields can be minimal or absent, primarily due to nucleic acid adsorption. The polyanionic property of nucleic acid is derived from the 5′-phosphate linkages and will support a large number of binding sites either directly with iron oxides or with multivalent cations bound to negatively charged clay particles. Therefore, it is reasonable to anticipate that longer nucleic acid polymers will have a larger number of ionic interactions with substrates carrying multiple positive charges, and thereby have increased binding strength with iron oxides. Other clay environments containing additional polyvalent cations associated with the negatively charged surface of clay particles can limit recovery as well.
The negative charge at the surface of clay particles can bind polyvalent cations and form an adsorptive bridge that binds nucleic acids. Carboxyl and hydroxyl groups of humic and fulvic acids form stable complexes with metal cations, with a binding strength order of Fe3+>Al3+>Pb2+>Ca2+>Mn2+>Mg2+. See Gu et al. Environmental Science & Technology (1994) 28: 38-46. A number of these metals, particularly Al3+, are prevalent in clay materials and can reduce microbial activity. See Wong et al., Microb. Ecol. (2004) 47: 80-86.
A common method for extraction of DNA and RNA from clay as well as more typical soils would allow exploration of vast environments that are considered recalcitrant to molecular microbiological analysis. The current state of the art can be exemplified by extraction procedures using low concentrations (100 mM) of phosphate in the extraction buffer only hoping to promote desorption. See Zhao, J., et al., Appl. Environ. Microbiol. (1996) 62: 316-322); Hurt et al., Appl. Environ. Microbiol. (2001) 67: 4495-4503); Andeer et al., Appl. Environ. Microbiol. (2012). Moreover, Direito et al. used 1 M sodium phosphate and heat (≧55° C.) to recover DNA from Mars analogue substrates by lysis using a bead milling process. See Direito, S. O., et al., FEMS Microbiol. Ecol. (2012) 81: 111-123. Unfortunately, heating a solution containing nucleic acids leads to the denaturing and fragmentation of the nucleic acids resulting in smaller nucleic acids that are less useful in sequencing protocols. Moreover, sheering of DNA using bead milling lysis techniques results in smaller fragments of DNA that limit the ability to analyze the species diversity in a sample due to the difficulties inherent in sequencing of small DNA fragments (<15 kb).
Taken together, iron cemented clays and soils with a high humic acid content are notoriously difficult to isolate nucleic acids from in high molecular weights amenable to sequencing efforts. Thus, the current disclosure overcomes this issue by prohibiting adsorption and maximizing desorption of nucleic acids from the surface of charged clay particles.